Computational protocol: Expression of the Arabidopsis thaliana BBX32 Gene in Soybean Increases Grain Yield

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Protocol publication

[…] Soy sequences having a B-box domain were collected from the GIS data base, comprising of gene predictions from Soybean genotype, Williams 82, whole genome sequence assembly. Of the initial ∼100 sequences collected, sequences representing pseudogenes and platz domain containing proteins were removed. In addition, 23 sequences that represented allelic/splice variants were separated and not included in the phylogenetic analysis. A total of 61 sequences representing the soy B-box genes were used to build the phylogenetic tree (). The soy sequences, along with 32 Arabidopsis B-box protein sequences () were aligned by ClustalW using MEGA program . [...] Tissue was sampled from plants grown at Jerseyville, Illinois at R5 stage at 5 timepoints; ZT 21 (3am), ZT 0 (6am), ZT 3 (9am), ZT 6 (noon), and ZT 9 (3pm). Sunrise occurred at 6:08am, sunset at 8:08pm. As each collection timepoint required 15 minutes to sample, we set the sampling that initiated at 6am at ZT 0. Three bioreps pooled from three plants from each entry were collected. 200 mg ground plant tissue was aliquoted to a 2.0 ml lysing matrix E tube from Q-biogen. Nucleic acids were isolated by the CTAB method and then precipitated overnight at −20°C in 800 µl 100 percent ethanol, 150 µl ammonium acetate and 3 µl glycogen. Pellets were washed 3× with 80 percent ethanol and resuspended in nuclease free water prior to DNase treatment for 1 h at 37°C. Total RNA was purified using the RNeasy kit from Qiagen according to the manufacturers' instructions. RNA yield was analyzed using a NanoDrop-1000 spectrophotometer and integrity by an Agilent 2100 Bioanalyzer. RNA amplification was performed according to the manufacturers' recommendations using the TargetAmp 1-round Biotin-aRNA amplification kit from EpiCentre. 12 µg of labeled RNA was then fragmented according to the standard protocols for gene expression analysis provided by Affymetrix. Fragmented cRNA samples were prepared and hybridized to custom GeneChips from Affymetrix according to the manufacturers' standard protocol.Signal intensities were normalized using RMA (Robust Multi-Array Algorithm) using Partek software (St. Louis, MO) and subsequently transformed into log2 scale. A fixed effect ANOVA analysis was done on the log transformed data using the PROC MIXED module of SAS (V9.1.3) to identify genes having significant differential expression between the transgenic events and the wildtype control at each time point. Estimates of the fold change differential between the average response of the two transgenic events and the WT event were calculated by that module. The raw p-values for the estimated fold changes were adjusted to correct for the multiple testing problem using SAS's PROC MULTTEST module with the FDR method of Benjamini and Hochberg. Those genes with a FDR adjusted p-value less than 0.05 and an estimated differential fold change greater than 2.0 are reflected in .To identify genes with a significant temporal oscillation profile, the PROC NLIN module of SAS was used to fit the log2 transformed intensity values to a sine wave model:where T is the time point (24 hour scale) at which each sample is taken, a, b, and c are the fitted parameters computed by the NLIN model, and k is fixed at 2π/24 to constrain the model to a 24 hour cycle. Parameter a represents the average intensity across all the time points, b represents the magnitude of the diurnal response (2b is the peak to trough range in intensity), and c is the time shift for the sine wave (c+6 is the peak time and c−6 is the trough time). The raw p-values for the sine wave model were adjusted for multiple testing. Those genes with the FDR adjusted p-value less than 0.01 were selected and the list was further filtered to include only those where parameter a (average intensity) was above a background level and parameter b (diurnal magnitude) was greater than 0.5 so that there would be at least a two-fold change from peak to trough.The microarray data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus 39 and are accessible through GEO Series accession number GSE30828 (). [...] The transcript abundance of the transgenes and miRNA targets in this paper were assayed by Taqman analysis (Applied Biosystems). Sequence specific TaqMan assays were designed using Primer Express 2.0 software, where primers and probes () were positioned at the polymorphic sites. Two TaqMan assays per target were run in duplex with internal control assay specific to 18S. All testing was done in ABI 7900HT real time cyclers. Assays detecting only the specific target, demonstrating efficiency of 90–110 percent and no endogenous control dependence were selected and tested for reproducibility. CV<2 percent was achieved at Log2 level over 3 log concentration variance, using synthetic controls. […]

Pipeline specifications

Software tools multtest, Primer Express
Databases GEO
Applications Gene expression microarray analysis, qPCR
Organisms Glycine max, Arabidopsis thaliana